The present invention generally relates to the use of HrmA proteins to elicit a hypersensitive response in plant cells, plant seeds, plant tissues and plants. The present invention also relates to the use of pathogen inducible or any promoters with the hrmA gene to express the HrmA proteins in plant cells, plant seeds, plant tissues and plants.
Plants are capable of combating disease at several levels. In many instances, defense responses are initiated by a specific gene-for-gene interaction, whereby the product of a particular plant defense gene interacts with a corresponding pathogen gene product (elicitor), thereby triggering a series of cellular events that culminate in a localized cell-death response (or hypersensitive response; Dangl et al., 1996; Gilchrist, 1998) and enhanced resistance in unchallenged parts of the plant (SAR; Ryals et al., 1996). The plant gene products involved in gene-for-gene defense mechanisms are typically receptor-like molecules, and the initial interactions of these putative receptors with their ligands (of pathogen origin) is thought to trigger a sophisticated series of signaling events (Bent, 1996; Baker et al., 1997). Among the consequences are increased local production of active oxygen species, accelerated local cell death, induction of salicylic acid and jasmonic acid synthesis, and production, in unchallenged parts of the plant, of a battery of proteins and metabolites that have been associated with enhanced systemic resistance to a broad range of pathogens (Dangl et al., 1996).
The properties of plants that are induced for SAR are attractive from the perspective of pathogen resistance: they are usually protected against a broad range of bacterial, fungal, and viral pathogens, yet they may display little or no harmful effects otherwise (e.g., serious yield losses, aberrant developmental patterns, etc.). As mentioned in the preceding paragraph, challenge with so-called incompatible pathogens, which necessarily leads to a hypersensitive response, induces SAR (Sticher et al., 1997). Challenge with non-pathogenic microbes can also induce SAR (Van Loon et al., 1998). Certain chemicals may be able to induce SAR in treated plants (Gorlach et al., 1996; Morris et al., 1998; Rao and Davis, 1999). The expression of any of a number of genes that, while not of pathogenic origin per se, can induce hypersensitive responses or cause disease-like lesions, can trigger SAR, apparently through a means similar to that by which incompatible pathogens induce SAR (Dangl et al., 1996).
In light of the range of stimuli known to induce SAR, several strategies have been tested to genetically engineer plants so that they are constitutive for SAR, or can be induced with agents not usually associated with disease and defense responses. Expression of both plant resistance and microbial avr genes in the same plant has been tested; when the avr gene is controlled by a promoter whose activity is induced upon challenge by pathogens (including those unrelated to the source of the avr gene), the resulting plants can respond to so-called compatible pathogens as if possessing a specific gene-for-gene system (Hammond-Kosack et al., 1994; 1998). Constitutive expression of genes whose products act downstream from the putative receptors can result in constitutive SAR (Oldroyd and Staskawicz, 1998). Interestingly, in some instances, the resulting plants displayed few (if any) detrimental side effects, indicating that it is possible to condition permanent SAR without seriously affecting plant growth and development, or crop yield (Bowling et al., 1997; Yu et al., 1998; Oldroyd and Staskawicz, 1998). Induced or constitutive expression of microbial avr gene, elicitor or elicitor-like genes and other so-called disease lesion-mimic genes can also induce SAR constitutively in plants (Dangl et al., 1996).
The hypersensitive response of higher plants is characterized by the rapid, localized death of plant cells at the site of pathogen invasion. It occurs during incompatible interactions, which typically involve a microorganism that causes disease only in another plant, and is associated with resistance against many nematodes, fungi, viruses, and bacteria. When HR is induced by a genetically engineered avr gene expressed under the control of a low-level expression promoter or other controlled expression promoters, the responses of the plant are subtle and, most likely, at a microscopic scale.
The avr genes from the species Pseudomonas syringae are suitable for the purpose of obtaining genetically engineered SAR. Different strains cause symptoms ranging from galls to xe2x80x9cwildfirexe2x80x9d blights, and well-characterized virulence (symptom enhancing) factors are as diverse as phytohormones and peptide toxins. Multiple patterns of host specificity (including, in some cases, avirulence (avr)-mediated gene-for-gene interactions) involve virtually all crop plants, and plant associations vary from epephytism to devastating pathogenesis. The interactions with diversified plant specises imply the possibility that avr genes may cause HR in many different plants.
It is now known that that elicitation of the HR by P. syringae requires a bacterium that is able to synthesize an Avr protein and to directly inject the Avr protein into the doomed plant cell. (See He, 1998, Ann. Rev. Phytopathol., 36:363-392). The ability of Pseudomonas syringae strains to elicit the HR or pathogenesis in nonhost or host plants, respectively, is controlled by the hrp genes, and typical Hrp mutants have the null phenotype of a nonpathogen in all plants. [See Proc. Nat""l. Acad. Sci. USA, 82:406 (1985); J. Bateriol., 168:512 (1986); and Mol. Plantxe2x80x94Microbe Interact., 4:132 (1991)]. Hrp genes are clustered, and some appear to be widely conserved in Gram-negative bacterial pathogens that cause eventual necrosis in their hosts. These pathogens include Pseudomonas syringae, Pseudomonas solancearum, Xanthomonas campestris, Erwinia amylovora, Erwinia stewartii, and Erwinia chrysanthemi. [See Mol. Plantxe2x80x94Microbe Interact., 5:390 (1992)]. The hrp clusters from Pseudomonas syringae pv. syringae 61 (which has been deposited with the American Type Culture Collection under the provisions of the Budapest Treaty and which is designated as ATCC 55427) encode for proteins that assemble the type III secretion system to deliver Avr protein into plant cells. Through genetic engineering, the avr gene can be expressed inside the plant cell, thus by passing the delivery system that is required in native bacterial system. As a result, the avr genes from P. syringae can thus be used to obtain even broad range protection in plants.
The present inventors have discovered that it is desirable to express a broad-spectrum avr gene that can elicit resistance response in many cultivars and plant species so that the same avr expression construct can be used to generate resistance in multiple plants and cultivars. Many avr genes are identified initially based on their ability to trigger the HR and resistance in one or a few cultivars of a given plant species (Leach and White, 1996). However, further examination of the avirulence effect of these avr genes on other plant species often uncovers additional plant species and cultivars that react with an HR to these avr genes. To date, more than 50 pathogen avr genes (most from bacteria) have been cloned and characterized. These avr genes provide a useful resource for genetic engineering of broad-spectrum resistance in many crop plants. The hrmA gene is a broad-spectrum avr gene; it has been shown to trigger an HR in all examined tobacco cultivars (Alfano et al., 1997) and transformed Arabidopsis thaliana (Q. Li and S. Shen, unpublished observation). The present inventors have also discovered that a pathogen-inducible plant promoter with a very low basal level of expression (estimated in between 10xe2x88x927-10xe2x88x924 of poly(A) RNA) can allow this strategy to work. The present inventors have demonstrated the use of the xcex940.3TobRB7 promoter sequence to express the highly potent HR elicitor gene, hrmA. However, a large collection of pathogen-infection-specific promoters can be used to make the inducible expression, or low-level non-inducible expression promoters can be used to make constitutive expression, of avr genes more versatile in different plants, tissues, and/or developmental stages. The expression of the avr genes, in particular example of this patent hrmA, would induce SAR thus warrant a new methods to genetically engineer SAR in crops.
The invention provides a method of increasing resistance of plants to pathogens that involves introducing avr genes into plants and expressing avr proteins.
The invention thus provides novel chimeric gene constructs that contain avr coding sequences.
The invention also provides transformed plant cells, plant seeds, plant tissues and transgenic plants transformed with novel chimeric gene constructs that contain the avr coding sequences.
The invention thus provides a method for the expression of the avr genes in plant cells, plant seeds, plant tissues and plants.
The invention also provides the use of pathogen inducible promoters, or any promoter to express the avr genes in plant cells, plant seeds, plant tissues and plants.
According to an embodiment of the invention, a bacterial HR elicitor, hrmA of P. syringae and homologeus thereof, are expressed in plants such as tobacco under the control of a promoter, such as xcex940.3TobRB7, which is expressed in low-level constitutively. A forced expression of the HR in otherwise susceptible plants leads to increased resistance to pathogen infection.
Additional advantages of the present invention will be set forth in the description and examples that follow, or may be learned from practicing the invention. These and other advantages may be realized and attained by means of the features, instrumentalities and/or combinations particularly described herein. It is also to be understood that the foregoing general description and the following detailed description are only exemplary and explanatory and are not to be viewed as limiting or restricting the invention as claimed.
The invention itself, together with further advantages, will best be understood by reference to the following detailed description taken in conjunction with the accompanying drawings.